Chromatography. Chromatography is a combination of two words; * Chromo Meaning color * Graphy representation of something on paper (writing)

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1 Chromatography Chromatography is a combination of two words; * Chromo Meaning color * Graphy representation of something on paper (writing)

2 Invention of Chromatography Mikhail Tswett invented chromatography in 1901 during his research on plant pigments. He used the technique to separate various plant pigments such as Chlorophylls, Xanthophylls and Carotenoids. Mikhail Tswett Russian Botanist ( )

3 Original Chromatography Experiment Start: A glass column is filled with powdered limestone (CaCO 3 ). An EtOH extract of leaf pigments is applied to the top of the column. EtOH is used to flush the pigments down the column. Later End: A series of colored bands is seen to form, corresponding to the different pigments in the original plant extract. These bands were later determined to be chlorophylls, xanthophylls and carotenoids.

4 Definition of chromatography It is a physical separation method of separation in which the components of a mixture are separated by differences in their distribution between two phases, one of which is stationary (stationary phase) while the other (mobile phase) moves through it in a definite direction. The substances must interact with the stationary phase to be retained and separated by it.

5 Definition of chromatography IUPAC definition : Chromatography is a physical method of separation in which the components to be separated are distributed between two phases, one of which is stationary while the other moves in a definite direction. The stationary phase may be a solid, or a liquid supported on a solid or gel, the mobile phase may be either a gas or a liquid.

6 Chromatography is used by scientists to: Mixture Separate Components Analyze Identify Purify Quantify Analyze examine a mixture, its components, and their relations to one another Identify determine the identity of a mixture or components based on known components Purify separate components in order to isolate one of interest for further study Quantify determine the amount of the a mixture and/or the components present in the sample

7 Chromatograph: Instrument employed for a chromatography. Eluent: Fluid entering a column. Eluate: Fluid exiting the column. Elution: The process of passing the mobile phase through the column. Flow rate: How much mobile phase passed / minute (ml/min). Linear velocity: Distance passed by mobile phase per 1 min in the column (cm/min).

8 Mobile Phase gas or liquid that carries the mixture of components through the stationary phase. Stationary Phase the part of the apparatus that holds the components as they move through it, separating them.

9 Real-life examples of uses for chromatography: Pharmaceutical Company Hospital Law Enforcement Environmental Agency Manufacturing Plant

10 Retention time: It is the characteristic time it takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions. Retardation factor (R): Fraction of an analyte in the mobile phase of a chromatographic system.

11 Liquid Chromatography separates liquid samples with a liquid solvent (mobile phase) and a column composed of solid beads (stationary phase) Gas Chromatography separates vaporized samples with a carrier gas (mobile phase) and a column composed of a liquid or of solid beads (stationary phase) Paper Chromatography separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip (stationary phase) Thin-Layer Chromatography separates dried liquid samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of alumina or silica gel (stationary phase)

13 Paper and Thin Layer Chromatography The solvent moves up paper by capillary action, carrying mixture components at different rates. solvent front Later solvent

14 Thin Layer Chromatography Here the mobile phase is a liquid Flowing past a thin layer of powder on a solid support. Substances that are less attracted to the solid or are more soluble in the liquid move faster. And so move further up the plate by the time that the process has been stopped by taking the plate out of the liqiud. - larger Rf

16 R f = distance moved by substance distance moved by solvent front For substances that are very soluble in the liquid R f will be close to... 1 For substances that are rather insoluble in the liquid R f will be close to... 0

17 How Does Chromatography Work? In all chromatographic separations, the sample is transported in a mobile phase. The mobile phase can be a gas, a liquid, or a supercritical fluid. The mobile phase is then forced through a stationary phase held in a column or on a solid surface. The stationary phase needs to be something that does not react with the mobile phase or the sample. The sample then has the opportunity to interact with the stationary phase as it moves past it. Samples that interact greatly, then appear to move more slowly. Samples that interact weakly, then appear to move more quickly. Because of this difference in rates, the samples can then be separated into their components.

18 Chromatography is based on a physical equilibrium that results when a solute is transferred between the mobile and a stationary phase. A A A A A K = distribution coefficient or partition ratio A A A A Cross Section of Equilibrium in a column. A are adsorbed to the stationary phase. A are traveling in the mobile phase. A A A K = C S C M Where C S is the molar concentration of the solute in the stationary phase and C M is the molar concentration in the mobile phase.

19 In a chromatography column, flowing gas or liquid continuously replaces saturated mobile phase and results in movement of A through the column. Flow Column is packed with particulate stationary phase. As a material travels through the column, it assumes a Gaussian concentration profile as it distributes between the stationary packing phase and the flowing mobile gas or liquid carrier phase.

21 In a mixture, each component has a different distribution coefficient, and thus spends a different amount of time absorbed on the solid packing phase vs being carried along with the flowing gas Flow Flow Flow Flow More volatile materials are carried through the column more rapidly than less volatile materials, which results in a separation.

23 The Elution of a Solute through a Chromatographic System Series of absorption-extraction process Equilibrium between the two phases The distribution system is continuously thermodynamically driven towards equilibrium Equilibrium process between two phases is complicated

Distribution of K.E. between both the phases Solute molecules leave the stationary phase when their K.E. is equal or greater than the P.E. of their interaction with the stationary phase No.

24 Distribution of K.E. between both the phases Solute molecules leave the stationary phase when their K.E. is equal or greater than the P.E. of their interaction with the stationary phase No.of molecules at the boundary (N 1 ) K.E. in excess of the P.E. Associated with molecular interaction with stationary phase (E A ) No.of molecules at the boundary (N 2 ) K.E. is less of the P.E. Associated with molecular interaction with stationary phase (E A ) At equilibrium N 1 = N 2

25 Elution Development in TLC Development of a Thin layer Plate

26 If a detector is used to determine when the components elute from the column, a series of Gaussian peaks are obtained, one for each component in the mixture that was separated by the column. Note: The first two components were not completely separated. Peaks in general tend to become shorter and wider with time.

27 The Theoretical Plate Theoretical plate is a term coined by Martin & Synge. It is based on a study in which they imagined that chromatographic columns were analogous to distillation columns and made up or numerous discrete but connected narrow layers or plates. Movement of the solute down the column then could be treated as a stepwise transfer. Theoretical plates (N) measure how efficiently a column can separate a mixture into its components. This efficiency is based on the retention time of the components and the width of the peaks.

28 N = Number of theoretical plates (a measure of efficiency) N = 16( t R w b ) 2 t R w b t R is the retention time; it is measured from the injection peak (or zero) to the intersection of the tangents. w b is the width of the base of the triangle; it is measured at the intersection of the tangents with the baseline.

29 N = 16( t R w b ) 2 t R t R w b Larger N w b Smaller N When the retention time, t R, is held constant, the column that produces peaks with narrower bases, w b, will be more efficient have a greater N value. Likewise a column that produces wider peaks will be less efficient have a smaller N value. This is because a smaller denominator, w b, will yield a larger overall number and a larger denominator will yield a smaller number.

30 Gas Chromatography Good for volatile samples (up to about 250 o C) microliter of liquid or 1-10 ml vapor Can detect <1 ppm with certain detectors Can be easily automated for injection and data analysis

31 Components of a Gas Chromatograph Gas Supply: (usually N 2 or He) Sample Injector: (syringe / septum) Column: 1/8 or 1/4 x 6-50 tubing packed with small uniform size, inert support coated with thin film of nonvolatile liquid Detector: TC - thermal conductivity FID - flame ionization detector

32 Gas Liquid Chromatography Here the mobile phase is an unreactive gas ( eg Nitrogen) flowing through a tube. And the stationary phase is an involatile liquid held on particles of a solid support.

33 In the animation below the red molecules are more soluble in the liquid (or less volatile) than are the green molecules.

34 In practice the Column is contained in a thermostatic oven. (Why?) About 1μL of liquid is injected into one end of the column. As each component reaches the other end it is detected and registered on a chart recorder. The Retention Time is characteristic of a particular substance. (for the same column, temperature, gas flow etc.) The area under each peak indicates the relative quantities.

35 Injection port Oven Recorder Detector Nitrogen cylinder Column

37 Chromatogram of petrol Suggest identities of some of the unlabelled peaks.

38 Schematic of a Commercial Gas Chromatograph

39 HP 5890 Capillary Gas Chromatograph with Robotic Sample Injector and Data Station

40 General Settings for GC Startup program Y axis: 1-5 v (0-1 volt is in bright light, 4-5 volt is dark) X axis: seconds

41 Plot of GC Elution Data for Dichloromethane and Chloroform On 25 cm Tide Column Good: Peaks are smooth, well separated and elute quickly

42 Plot of GC Elution Data for Dichloromethane and Chloroform On 25 cm Tide Column Poor: peaks are noisy, due to flickering flame, and elute slowly. To fix: Adjust sensor so that it is looking at the blue portion of the flame. (Verify the flame is blue.)

43 Determination of the Amount of Sample Components Present The peak height is proportional to the amount of material eluting from the column at any given time, The area under the peak is a measure of the total amount of material that has eluted from the column. Electronic integrators are used for area measurement in commercial GCs. We will be using ALGEBRA.

44 The Gaussian curve can be approximated as triangular in shape, to simplify area measurement. Area = 1/2 w b h h w b NOTE: the height is measured to the top of the tangents, which is above the actual curve peak.

45 GEL FILTRATION Gel filtration separates molecules according to the differences in size as they pass through the filtration medium packed in the column. It is well suited for biomolecules that are sensitive to ph,concentration and harsh environment. Parameters that affects gel filtration are, particle size, flow rate, packaging density, porosity of the particle and viscosity of the mobile phase.

47 MATERIALS REQUIRED Cross linked dextrans (sephadex) Agarose (sepharose) Polyacrylamide Porous glass gel. APPLICATIONS Fractionation (purification of the desired protein using suitable gel) Molecular weight determination

48 ION EXCHANGE Ion exchange chromatography is used to remove ions of one type from a mixture and replace them by ions of another type. The basic principle is reversible competitive binding

49 ION EXCHANGERS Cation exchangers (negative ions stationary) Anion exchangers (positive ions - stationary) Four types of polymers are commonly used. They are, Synthetic hydrophobic polymer resins crosslinked with divinylbenzene. Naturally occuring as well as synthetic polymers(cellulose) Synthetic hydrophilic polymers Silica gel

50 AFFINITY CHROMATOGRAPHY Affinity chromatography includes bioaffinity, dye-ligand affinity and immobilized metal ion afffinity techniques. It is based on the formation of the specific and reversible complexes between a pair of biomolecules.

51 HPLC HPLC is a physical separation technique in which a sample dissolved in a liquid is injected into a column packed with small particles and it is separated into its constituent components HPLC is probably the most important and widely used analytical technique for quantitative analysis of organics and biomolecules HPLC is applicable to many kind of samples: Most useful for pharmaceuticals, biomolecules, and labile organics

53 HPLC Instrumentation Overview Principle Pattern An Example Solvent Reservoirs Controller Solvent Cabinet Vacuum Degasser Binary Pump Autosampler Thermostatted Column Compartment Detector 53

55 Typical HPLC Unit

57 HPTLC HPTLC is a sophisticated form of TLC. Fastest of all chromatographic techniques. Any combinations of stationary and mobile phases can be used. Analytical HPTLC is used for micro preparative analysis (ie., separation of milligram scale for analysis of fraction ) Gives more sharper and compact bands with minimum distance of migration. Used for both qualitative and quantitative analysis.

58 Applications of HPLC Techniques A mixture of sugars containing fructose, sucrose, lactose, maltose have been separated on column Zorbax NH2 with 70% acetonitrile in aq. Phase with RI detector In environmental pollution analysis for ions in trace concentrations For Bioseparation, DNA, carbohydrates, lipids, proteins and amino acids Rest for assignment..

Chromatography. writing in color

Chromatography. writing in color Chromatography writing in color Outlines of Lecture Chromatographic analysis» Principles and theory.» Definition.» Mechanism.» Types of chromatography.» Uses of Chromatography. In 1906 Mikhail Tswett used